PROJECT SUMMARY/ABSTRACT The mediodorsal thalamus (MD) is suggested to play a critical role in cognition through its extensive glutamatergic innervation of the medial prefrontal cortex (mPFC). These MD terminals provide AMPA-mediated excitation to both excitatory pyramidal neurons and gamma-aminobutyric (GABA)-ergic inhibitory interneurons. GABAergic interneurons, particularly the parvalbumin (PV)-expressing fast-spiking subtype, are fundamental in regulating the output of their neighboring pyramidal neurons, maintaining the excitation/inhibition (E/I) balance, and coordinating synchronous activity in populations of neurons. Interestingly, this neuronal subtype receives MD inputs, and their activity has been linked to working memory, attention, and executive functions associated with the PFC. However, how inhibitory interneurons are modulated by MD thalamocortical input remains unknown. We hypothesize that MD afferents drive mPFC inhibitory activity, aiding in the regulation of local circuit activity and thus optimizing performance on mPFC dependent tasks. This is based on feedforward inhibition observed not only in primary thalamic relays, but also recent evidence of this phenomenon in the anterior cingulate cortex. Given this, we propose that downregulation of MD activity will impair GABAergic activity in the prelimbic mPFC, causing dysregulation of local circuit activity, and cognitive deficits. To explore this possibility, we utilize Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that allow us to selectively target glutamatergic MD neurons, and transiently inhibit their activity in adult rats by expression of the inhibitory hM4D receptor. Using pharmacogenetics, we can evaluate the consequences of MD inhibition by two primary methodologies. With the designer ligand, Clozapine-N-Oxide (CNO), we will treat animals systemically and explore the effects of MD inhibition on mPFC-dependent behaviors using a working memory and cognitive flexibility task. We will also take slices containing the mPFC and MD afferents, bath apply CNO, and determine resultant changes in the circuitry using whole cell-recordings. Thus far, we have successfully demonstrated that MD inhibition resulted in a significant decrease of GABAAR-mediated IPSCs in PFC pyramidal neurons. This loss in IPSCs appears to be greater at ?1- GABAARs. Further, these decreases of GABA activity in the PFC are associated with cognitive dysfunction in our MD-inhibited animals. Using a T- maze delayed-alternation task, we have shown that MD inhibition impairs working memory performance, which can be rescued by treatment with the GABAergic ?1-positive allosteric modulator, indiplon. These findings raise intriguing questions of how MD inhibition mechanistically influences mPFC local circuitry, and whether the MD regulates the mPFC through PV interneurons to impact cognition. We will further address these questions using a combination of surgical, electrophysiological, behavioral, and pharmacogenetic techniques.